Interest in the administration of both therapeutic and antigenic proteins and peptides has grown considerably in recent years due to improvements in the quality and quantity of recombinant proteins and synthetic peptides now available. These molecules, however, suffer the disadvantage of having short biological half lives following parenteral administration and are degraded in the intestine following oral administration. Furthermore, if orally or nasally administered, these molecules show poor absorption through the mucous membranes.
Biodegradable polymers such as polylactide-co-glycolides (PLG) have been used to encapsulate proteins and peptides and other drugs for parenteral and/or oral delivery in order to try to achieve a stable and therapeutically adequate level of drug over an extended period of time. Previous investigators have claimed that antigenic protein and peptides can be encapsulated in microcapsules to deliver "pulses" (i.e. "intermittent doses") of antigenic material for the development of vaccines (see e.g. U.S. Pat. No. 5,075,109 to Tice et al.). The use of microencapsulation to protect sensitive bioactive agents against degradation is well known in the art, however, the use of biodegradable microparticles in controlled release delivery systems seldom results in satisfactory release profiles.
The drug release pattern for a microcapsule is dependent upon numerous factors. For example, the type of drug encapsulated and the form in which it is present (i.e. liquid or powder) may affect the drugs release pattern. Another factor which may affect the drug release pattern is the type of polymer used to encapsulate the drug. Other factors affecting the drug release pattern include the drug loading, the manner of distribution in the polymer, the particle size and the particle shape.
There are several methods known for the production of microparticles. Typical methods for producing microparticles include solvent evaporation and phase separation. With production methods such as solvent evaporation, as much as 50% w/w of insoluble or poorly soluble materials, may be incorporated in biodegradable microparticles. However, with more water soluble materials, such as peptides, drug loadings have generally been much lower.
Consequently, the use of phase separation for production of microparticles may be better suited for the formulation of microparticles containing more water soluble compounds. Phase separation methods of microparticle preparation allow a more efficient incorporation of drugs and can easily be scaled up for industrial purposes. The process of phase separation usually employs an emulsion or a suspension of the drug particles in a solution of a high molecular weight polymer and an organic polymer solvent. A non-solvent is then added to the suspension or emulsion, causing the polymer to separate from solution and to encapsulate the suspended drug particles or droplets containing them. The resulting microparticles (which are still swollen with solvent) are then normally hardened by a further addition of a non-solvent or by some other process which strengthens and improves the properties of the microparticles.
A variety of techniques to produce microparticles have been described in the prior art. For example, United Kingdom Patent Application No. 2,234,896 to Bodmer et al. describes a method of forming microparticles by mixing a solution of the polymer dissolved in an appropriate solvent with a solution of a drug. Microparticle formation is then induced by the addition of a phase inducing agent. European Patent Application 0 330 180 to Hyon et al. describes a process for preparing polylactic acid-type microparticles by adding a solution of a drug and a polymer in a mixed solvent to a phase inducing agent and evaporating the original solvent microparticle formation. Other examples of processes for preparing microparticles by phase separation technique have been described in U.S. Pat. Nos. 4,732,763 to Beck et al. and 4,897,268 to Tice et al. and by Ruiz et al. in the International Journal of Pharmaceutics (1989) 49:69-77 and in Pharmaceutical Research (1990) 9:928-934.
Despite numerous modifications to the process of polylactide-co-glycolides microparticle formation by phase separation, several problems are usually encountered when following the described techniques of microencapsulation. Such problems include: low or negligible and inefficient drug entrapment (&lt;0.5% w.w), aggregation of particles, formation of non-spherical particles, formation of particles with surfaces that are not smooth and which have defects, the presence of large particles with a wide range of sizes (5 .mu.m-250 .mu.m) and the presence of non-particulate material. All these problems reduce the effectiveness and reproducability of the microparticles produced by these methods for use in controlled release delivery systems.
Traditional immunization schedules require a primary and one or more booster immunizations to achieve protective immunity. Many individuals, however, fail to receive the necessity booster immunization and therefore, fail to adequately protect themselves against the respective disease. Furthermore, this immunization regimen fails to provide a continuous dose response, leaving an individual move susceptible to diseases at one time point compared to another. Traditional immunization regimens provide an antigen to the immune system in discrete pulses. Previous investigators have attempted to convert multiple dose immunization schedules to single dose schedules using controlled release antigen delivery systems comprising biodegradable microcapsules. For example, U.S. Pat. No. 5,075,109 to Tice et al. describes a method of immunization in which the antigen is delivered in microcapsules of different sizes to attempt to provide an initial dose response followed by a subsequent dose response. The method of Tice attempts to mimic the traditional immunization regime using a single dose of the requisite antigen. Although this method alleviates the necessity for providing a booster immunization, this method does not provide a continuous administration of antigen and simply provides the traditional burst of antigen regimen.
The theory of providing continuous dose response of an antigen to elicit a prolonged immune response was discussed in 1987 by Wise et al. in Advanced Drug Delivery Reviews (1987) 1:19-39. Wise et al. stated that if an antigen was released in a continuous manner, the amount of antigen presented to the immune system would be too low to induce a protective immune response and may actually lead to tolerance. Recently, Walker in Vaccine (1994) 5:387-400 similarly stated that a sustained release of small amounts of antigen over a prolonged time period would likely induce tolerance rather than provide an effective immune response to the antigen.
The present invention solves many of the problems associated with current immunization methods. In particular, contrary to the teachings in the prior art, the present invention provides an essentially continuous release of an antigen from microparticles prepared using the novel method described by the present invention. It has been surprisingly discovered in accordance with the present invention that a continuous release of antigen results in the induction of immune responses which are comparable to those induced by the potent immunological adjuvant, aluminum hydroxide.